Fusion proteins comprising enzyme replacement therapy enzymes

Abstract

Provided herein are fusion proteins that comprise an enzyme replacement therapy enzyme and an Fc region, as well as methods of using such proteins to treat a lysosomal storage disorder. Methods for transporting agents across the blood-brain barrier are also provided herein.

What is claimed is: 
     
         1 . A protein comprising:
 (a) a first Fc polypeptide that is linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and   (b) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the first Fc polypeptide and/or the second Fc polypeptide does not include an immunoglobulin heavy and/or light chain variable region sequence or an antigen-binding portion thereof.   
     
     
         2 . The protein of  claim 1 , wherein the ERT enzyme is iduronate 2-sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof. 
     
     
         3 . The protein of  claim 2 , wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of any one of SEQ ID NOS:91, 92, 114, 230, and 234. 
     
     
         4 . The protein of  claim 3 , wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NOS:91, 92, 114, 230, and 234. 
     
     
         5 . The protein of  claim 1 , wherein the ERT enzyme is N-sulfoglucosamine sulfohydrolase (SGSH), an SGSH variant, or a catalytically active fragment thereof. 
     
     
         6 . The protein of  claim 5 , wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of any one of SEQ ID NOS:119 and 120. 
     
     
         7 . The protein of  claim 6 , wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NOS:119 and 120. 
     
     
         8 . The protein of  claim 1 , wherein the ERT enzyme is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof. 
     
     
         9 . The protein of  claim 8 , wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of any one of SEQ ID NOS:121, 122, and 123. 
     
     
         10 . The protein of  claim 9 , wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NOS:121, 122, and 123. 
     
     
         11 . The protein of  claim 1 , wherein the ERT enzyme is β-glucocerebrosidase (GBA), a GBA variant, or a catalytically active fragment thereof. 
     
     
         12 . The protein of  claim 11 , wherein the ERT enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of any one of SEQ ID NOS:93 and 94. 
     
     
         13 . The protein of  claim 12 , wherein the ERT enzyme comprises the amino acid sequence of any one of SEQ ID NOS:93 and 94. 
     
     
         14 . The protein of any one of  claims 1  to  13 , wherein the first Fc polypeptide is a fusion polypeptide that is linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or by a polypeptide linker. 
     
     
         15 . The protein of  claim 14 , wherein the polypeptide linker is a flexible polypeptide linker. 
     
     
         16 . The protein of  claim 15 , wherein the flexible polypeptide linker is a glycine-rich linker. 
     
     
         17 . The protein of  claim 16 , wherein the glycine-rich linker is G 4 S (SEQ ID NO:239) or (G 4 S) 2  (SEQ ID NO:240). 
     
     
         18 . The protein of any one of  claims 1  to  17 , wherein the second Fc polypeptide is linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. 
     
     
         19 . The protein of  claim 18 , wherein the second Fc polypeptide is a fusion polypeptide that is linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or by a polypeptide linker. 
     
     
         20 . The protein of  claim 18  or  19 , wherein the N-terminus of the first Fc polypeptide and/or the N-terminus of the second Fc polypeptide is linked to the ERT enzyme. 
     
     
         21 . The protein of  claim 20 , wherein the N-terminus of the first Fc polypeptide is linked to one ERT enzyme and the N-terminus of the second Fc polypeptide is linked to the other ERT enzyme. 
     
     
         22 . The protein of  claim 18  or  19 , wherein the C-terminus of the first Fc polypeptide and/or the C-terminus of the second Fc polypeptide is linked to the ERT enzyme. 
     
     
         23 . The protein of  claim 22 , wherein the C-terminus of the first Fc polypeptide is linked to one ERT enzyme and the C-terminus of the second Fc polypeptide is linked to the other ERT enzyme. 
     
     
         24 . The protein of  claim 18  or  19 , wherein the N-terminus of the first Fc polypeptide is linked to one ERT enzyme and the C-terminus of the second Fc polypeptide is linked to the other ERT enzyme. 
     
     
         25 . The protein of  claim 18  or  19 , wherein the C-terminus of the first Fc polypeptide is linked to one ERT enzyme and the N-terminus of the second Fc polypeptide is linked to the other ERT enzyme. 
     
     
         26 . The protein of any one of  claims 1  to  17 , wherein the protein comprises a single ERT enzyme, and wherein the N-terminus or the C-terminus of the first Fc polypeptide is linked to the ERT enzyme. 
     
     
         27 . The protein of any one of  claims 18  to  25 , wherein the protein comprises two ERT enzymes. 
     
     
         28 . The protein of any one of  claims 14  to  17 , wherein the fusion polypeptide comprises from N- to C-terminus: the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof; the polypeptide linker; and the first Fc polypeptide. 
     
     
         29 . The protein of any one of  claims 1  to  28 , wherein the first Fc polypeptide is a modified Fc polypeptide and/or the second Fc polypeptide is a modified Fc polypeptide. 
     
     
         30 . The protein of  claim 29 , wherein the first Fc polypeptide and the second Fc polypeptide each contain modifications that promote heterodimerization. 
     
     
         31 . The protein of  claim 30 , wherein the Fc dimer is an Fc heterodimer. 
     
     
         32 . The protein of  claim 30  or  31 , wherein one of the Fc polypeptides has a T366W substitution and the other Fc polypeptide has T366S, L368A, and Y407V substitutions, according to EU numbering. 
     
     
         33 . The protein of  claim 32 , wherein the first Fc polypeptide contains the T366S, L368A, and Y407V substitutions and the second Fc polypeptide contains the T366W substitution. 
     
     
         34 . The protein of  claim 33 , wherein the first Fc polypeptide is linked to the ERT enzyme and comprises the amino acid sequence of any one of SEQ ID NOS:117, 232, and 236. 
     
     
         35 . The protein of  claim 32 , wherein the first Fc polypeptide contains the T366W substitution and the second Fc polypeptide contains the T366S, L368A, and Y407V substitutions. 
     
     
         36 . The protein of  claim 35 , wherein the first Fc polypeptide is linked to the ERT enzyme and comprises the amino acid sequence of any one of SEQ ID NOS:118, 233, and 237. 
     
     
         37 . The protein of any one of  claims 29  to  36 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises a native FcRn binding site. 
     
     
         38 . The protein of any one of  claims 29  to  37 , wherein the first Fc polypeptide and the second Fc polypeptide do not have effector function. 
     
     
         39 . The protein of any one of  claims 29  to  37 , wherein the first Fc polypeptide and/or the second Fc polypeptide includes a modification that reduces effector function. 
     
     
         40 . The protein of  claim 39 , wherein the modification that reduces effector function is the substitutions of Ala at position 234 and Ala at position 235, according to EU numbering. 
     
     
         41 . The protein of  claim 40 , wherein the first Fc polypeptide is linked to the ERT enzyme and comprises the amino acid sequence of any one of SEQ ID NOS:115, 231, and 235. 
     
     
         42 . The protein of  claim 40 , wherein the first Fc polypeptide is linked to the ERT enzyme and comprises the amino acid sequence of any one of SEQ ID NOS:149, 150, 152, and 153. 
     
     
         43 . The protein of any one of  claims 29  to  42 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises amino acid changes relative to the native Fc sequence that extend serum half-life. 
     
     
         44 . The protein of  claim 43 , wherein the amino acid changes comprise substitutions of Tyr at position 252, Thr at position 254, and Glu at position 256, according to EU numbering. 
     
     
         45 . The protein of  claim 43 , wherein the amino acid changes comprise substitutions of Leu at position 428 and Ser at position 434, according to EU numbering. 
     
     
         46 . The protein of  claim 43 , wherein the amino acid changes comprise a substitution of Ser or Ala at position 434, according to EU numbering. 
     
     
         47 . The protein of any one of  claims 29  to  46 , wherein the first Fc polypeptide and/or the second Fc polypeptide specifically binds to a transferrin receptor (TfR). 
     
     
         48 . The protein of  claim 47 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises at least two substitutions at positions selected from the group consisting of 384, 386, 387, 388, 389, 390, 413, 416, and 421, according to EU numbering. 
     
     
         49 . The protein of  claim 48 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises at least three, four, five, six, seven, eight, or nine substitutions at the positions. 
     
     
         50 . The protein of  claim 48  or  49 , wherein the first Fc polypeptide and/or the second Fc polypeptide further comprises one, two, three, or four substitutions at positions comprising 380, 391, 392, and 415, according to EU numbering. 
     
     
         51 . The protein of any one of  claims 48  to  50 , wherein the first Fc polypeptide and/or the second Fc polypeptide further comprises one, two, or three substitutions at positions comprising 414, 424, and 426, according to EU numbering. 
     
     
         52 . The protein of any one of  claims 48  to  51 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises Trp at position 388. 
     
     
         53 . The protein of any one of  claims 48  to  52 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises an aromatic amino acid at position 421. 
     
     
         54 . The protein of  claim 53 , wherein the aromatic amino acid at position 421 is Trp or Phe. 
     
     
         55 . The protein of any one of  claims 48  to  54 , wherein 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. 
     
     
         56 . The protein of  claim 55 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from the following: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe. 
     
     
         57 . The protein of  claim 56 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises 11 positions as follows: 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. 
     
     
         58 . The protein of  claim 56  or  57 , wherein the first Fc polypeptide and/or the second Fc polypeptide has a CH3 domain with at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOS:34-38, 58, 60-90, 151, and 156-229. 
     
     
         59 . The protein of  claim 58 , wherein the residues at at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions corresponding to EU index positions 380, 384, 386, 387, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424 and 426 of any one of SEQ ID NOS:34-38, 58, 60-90, 151, and 156-229 are not deleted or substituted. 
     
     
         60 . The protein of  claim 58  or  59 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:156-229. 
     
     
         61 . The protein of  claim 60 , wherein the first Fc polypeptide and/or the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:157, 169, 181, 193, 205, and 217. 
     
     
         62 . The protein of  claim 60 , wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:115, 231, and 235, and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:205 and 228. 
     
     
         63 . The protein of  claim 60 , wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:115, 231, and 235, and the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:169 and 229. 
     
     
         64 . The protein of any one of  claims 47  to  63 , wherein the first Fc polypeptide and/or the second Fc polypeptide binds to the apical domain of the TfR. 
     
     
         65 . The protein of any one of  claims 47  to  64 , wherein the binding of the protein to the TfR does not substantially inhibit binding of transferrin to the TfR. 
     
     
         66 . The protein of any one of  claims 47  to  65 , wherein the first Fc polypeptide and/or the second Fc polypeptide has an amino acid sequence identity of at least 75%, or at least 80%, 85%, 90%, 92%, or 95%, as compared to the corresponding wild-type Fc polypeptide. 
     
     
         67 . The protein of  claim 66 , wherein the corresponding wild-type Fc polypeptide is a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide. 
     
     
         68 . The protein of any one of  claims 47  to  67 , wherein uptake of the ERT enzyme into the brain is at least ten-fold greater as compared to the uptake of the ERT enzyme in the absence of the first Fc polypeptide and/or the second Fc polypeptide or as compared to the uptake of the ERT enzyme without the modifications to the first Fc polypeptide and/or the second Fc polypeptide that result in TfR binding. 
     
     
         69 . The protein of any one of  claims 1  to  68 , wherein the first Fc polypeptide is not modified to bind to a blood-brain barrier (BBB) receptor and the second Fc polypeptide is modified to specifically bind to a TfR. 
     
     
         70 . The protein of any one of  claims 1  to  68 , wherein the first Fc polypeptide is modified to specifically bind to a TfR and the second Fc polypeptide is not modified to bind to a BBB receptor. 
     
     
         71 . The protein of any one of  claims 1  to  70 , wherein the protein does not include an immunoglobulin heavy and/or light chain variable region sequence or an antigen-binding portion thereof. 
     
     
         72 . A polypeptide comprising an Fc polypeptide that is linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, wherein the Fc polypeptide contains one or more modifications that promote its heterodimerization to another Fc polypeptide. 
     
     
         73 . The polypeptide of  claim 72 , wherein the ERT enzyme is iduronate 2-sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof. 
     
     
         74 . The polypeptide of  claim 72 , wherein the ERT enzyme is N-sulfoglucosamine sulfohydrolase (SGSH), an SGSH variant, or a catalytically active fragment thereof. 
     
     
         75 . The polypeptide of  claim 72 , wherein the ERT enzyme is acid sphingomyelinase (ASM), an ASM variant, or a catalytically active fragment thereof. 
     
     
         76 . The polypeptide of  claim 72 , wherein the ERT enzyme is β-glucocerebrosidase (GBA), a GBA variant, or a catalytically active fragment thereof. 
     
     
         77 . The polypeptide of any one of  claims 72  to  76 , wherein the Fc polypeptide is a fusion polypeptide that is linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or by a polypeptide linker. 
     
     
         78 . The polypeptide of  claim 77 , wherein the fusion polypeptide comprises from N- to C-terminus: the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof; the polypeptide linker; and the first Fc polypeptide. 
     
     
         79 . The polypeptide of any one of  claims 72  to  78 , wherein the Fc polypeptide contains T366S, L368A, and Y407V substitutions, according to EU numbering. 
     
     
         80 . The polypeptide of  claim 79 , wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:115, 117, 231, 232, 235, and 236. 
     
     
         81 . The polypeptide of  claim 79 , wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:149 and 150. 
     
     
         82 . The polypeptide of any one of  claims 72  to  78 , wherein the Fc polypeptide contains a T366W substitution. 
     
     
         83 . The polypeptide of  claim 82 , wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:118, 233, and 237. 
     
     
         84 . The polypeptide of  claim 82 , wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:152-155. 
     
     
         85 . The polypeptide of any one of  claims 72  to  84 , wherein the polypeptide further comprises the other Fc polypeptide. 
     
     
         86 . A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of  claims 72  to  84 . 
     
     
         87 . A vector comprising the polynucleotide of  claim 86 . 
     
     
         88 . A host cell comprising the polynucleotide of  claim 86  or the vector of  claim 87 . 
     
     
         89 . The host cell of  claim 88 , further comprising a polynucleotide comprising a nucleic acid sequence encoding the other Fc polypeptide. 
     
     
         90 . A method for producing a polypeptide comprising an Fc polypeptide that is linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof, comprising culturing a host cell under conditions in which the polypeptide encoded by the polynucleotide of  claim 86  is expressed. 
     
     
         91 . A method of treating a lysosomal storage disorder (LSD), the method comprising administering the protein of any one of  claims 1  to  71  or the polypeptide of any one of  claims 72  to  85  to a patient in need thereof. 
     
     
         92 . A method of decreasing the accumulation of a toxic metabolic product in a patient having an LSD, the method comprising administering the protein of any one of  claims 1  to  71  or the polypeptide of any one of  claims 72  to  85  to the patient. 
     
     
         93 . The method of  claim 91  or  92 , wherein the LSD is Hunter syndrome, and the ERT enzyme is IDS. 
     
     
         94 . The method of  claim 93 , wherein the toxic metabolic product comprises heparin sulfate-derived disaccharides and/or dermatan sulfate-derived disaccharides. 
     
     
         95 . The method of  claim 91  or  92 , wherein the LSD is Sanfilippo syndrome A, and the ERT enzyme is SGSH. 
     
     
         96 . The method of  claim 95 , wherein the toxic metabolic product comprises heparan sulfate-derived oligosaccharides. 
     
     
         97 . The method of  claim 91  or  92 , wherein the LSD is Niemann-Pick disease, and the ERT enzyme is ASM. 
     
     
         98 . The method of  claim 97 , wherein the toxic metabolic product comprises sphingomyelin. 
     
     
         99 . The method of  claim 91  or  92 , wherein the LSD is Gaucher's disease or Parkinson's disease, and the ERT enzyme is GBA. 
     
     
         100 . The method of  claim 99 , wherein the toxic metabolic product comprises glucosylceramide. 
     
     
         101 . A pharmaceutical composition comprising the protein of any one of  claims 1  to  71  or the polypeptide of any one of  claims 72  to  85  and a pharmaceutically acceptable carrier. 
     
     
         102 . A method of monitoring substrate accumulation to assess IDS activity, the method comprising:
 (a) disrupting cells in a cell or tissue sample from a subject administered the protein of any one of  claims 2  to  4 ,  14  to  41 , and  43  to  71  or the polypeptide of any one of  claims 72  to  73 ,  77  to  80 ,  82  to  83 , and  85  to break open microvesicles to obtain a glycosaminoglycan (GAG) solution to be analyzed;   (b) digesting the GAG solution with at least one heparinase and chrondroitinase B to obtain GAG-derived disaccharides;   (c) analyzing the GAG-derived disaccharides by mass spectrometry; and   (d) determining the levels of heparan sulfate- and/or dermatan sulfate-derived disaccharides, wherein decreased levels of heparan sulfate- and/or dermatan sulfate-derived disaccharides compared to a control that lacks IDS activity is indicative of increased IDS activity in the sample compared to the control.   
     
     
         103 . The method of  claim 102 , wherein the step of disrupting cells comprises at least one freeze-thaw cycle and at least one sonication step. 
     
     
         104 . The method of  claim 103 , wherein the cells are from a tissue sample and the method comprises at least three, four, or five freeze-thaw cycles. 
     
     
         105 . The method of any one of  claims 102  to  104 , wherein the subject is a mouse deficient in IDS activity. 
     
     
         106 . The method of any one of  claims 102  to  104 , wherein the subject is a non-human primate. 
     
     
         107 . The method of any one of  claims 102  to  104 , wherein the subject is a human patient having Hunter syndrome. 
     
     
         108 . A method of monitoring substrate accumulation to assess SGSH activity, the method comprising:
 (a) disrupting cells in a cell or tissue sample from a subject administered the protein of any one of  claims 5  to  7 ,  14  to  33 ,  35 ,  37  to  40 , and  42  to  71  or the polypeptide of any one of  claims 72 ,  74 ,  77  to  79 ,  81  to  82 , and  84  to  85  to break open microvesicles to obtain a glycosaminoglycan (GAG) solution to be analyzed;   (b) digesting the GAG solution with at least one heparinase to obtain GAG-derived disaccharides;   (c) analyzing the GAG-derived disaccharides by mass spectrometry; and   (d) determining the levels of heparan sulfate-derived disaccharides, wherein decreased levels of heparan sulfate-derived disaccharides compared to a control that lacks SGSH activity is indicative of increased SGSH activity in the sample compared to the control.   
     
     
         109 . The method of  claim 108 , wherein the step of disrupting cells comprises at least one freeze-thaw cycle and at least one sonication step. 
     
     
         110 . The method of  claim 109 , wherein the cells are from a tissue sample and the method comprises at least three, four, or five freeze-thaw cycles. 
     
     
         111 . The method of any one of  claims 108  to  110 , wherein the subject is a mouse deficient in SGSH activity. 
     
     
         112 . The method of any one of  claims 108  to  110 , wherein the subject is a non-human primate. 
     
     
         113 . The method of any one of  claims 108  to  110 , wherein the subject is a human patient having Sanfilippo syndrome A. 
     
     
         114 . A method for transporting an agent across the BBB of a mammal, comprising exposing the BBB to a protein that binds to a TfR with an affinity of from about 50 nM to about 250 nM, wherein the protein is linked to the agent and transports the linked agent across the BBB. 
     
     
         115 . The method of  claim 114 , wherein the maximum concentration (C max ) of the agent in the brain of the mammal is improved. 
     
     
         116 . The method of  claim 114  or  115 , wherein the agent is useful for treating an LSD. 
     
     
         117 . A method for treating an LSD, comprising administering to a mammal a protein that binds to a TfR with an affinity of from about 50 nM to about 250 nM, wherein the protein is linked to an agent for treating the LSD, thereby exposing the brain of the mammal to the agent. 
     
     
         118 . The method of any one of  claims 114  to  117 , wherein the protein improves the C max  of the agent in the brain as compared to the agent linked to a reference protein that binds to the TfR with a weaker affinity. 
     
     
         119 . The method of any one of  claims 114  to  118 , wherein the protein improves the C max  of the agent at a therapeutically effective concentration in the mammal as compared to the agent linked to a reference protein that binds to the TfR with a weaker affinity. 
     
     
         120 . The method of any one of  claims 118  to  119 , wherein the reference protein binds to the TfR with an affinity of about 600 nM, or weaker. 
     
     
         121 . The method of any one of  claims 114  to  120 , wherein the TfR is a primate TfR. 
     
     
         122 . The method of  claim 121 , wherein the primate TfR is a human TfR. 
     
     
         123 . The method of any one of  claims 114  to  122 , wherein the protein binds to the TfR apical domain. 
     
     
         124 . The method of any one of  claims 114  to  123 , wherein the protein binds to the TfR with an affinity of from about 100 nM to about 200 nM. 
     
     
         125 . The method of any one of  claims 114  to  124 , wherein the protein binds to the TfR with an affinity of from about 110 nM to about 150 nM. 
     
     
         126 . The method of any one of  claims 119  to  125 , wherein the therapeutically effective concentration of the agent is a concentration that treats one or more symptoms of an LSD in the mammal. 
     
     
         127 . The method of any one of  claims 114  to  126 , wherein the agent is a protein replacement therapeutic. 
     
     
         128 . The method of any one of  claims 114  to  127 , wherein the agent or protein replacement therapeutic is an enzyme. 
     
     
         129 . The method of  claim 128 , wherein the enzyme decreases the accumulation of a toxic metabolic product in the brain of the mammal having the LSD to a greater extent when linked to the protein as compared to when the enzyme is linked to the reference protein. 
     
     
         130 . The method of  claim 128  or  129 , wherein the enzyme is IDS and the LSD is Hunter syndrome. 
     
     
         131 . The method of  claim 130 , wherein the toxic metabolic product comprises heparin sulfate-derived disaccharides and/or dermatan sulfate-derived disaccharides. 
     
     
         132 . The method of  claim 128  or  129 , wherein the enzyme is SGSH and the LSD is Sanfilippo syndrome A. 
     
     
         133 . The method of  claim 128  or  129 , wherein the enzyme is ASM and the LSD is Niemann-Pick disease. 
     
     
         134 . The method of  claim 128  or  129 , wherein the enzyme is GBA and the LSD is Gaucher's disease. 
     
     
         135 . The method of any one of  claims 114  to  126 , wherein the agent comprises an antibody variable region. 
     
     
         136 . The method of  claim 135 , wherein the agent comprises an antibody fragment. 
     
     
         137 . The method of  claim 136 , wherein the agent comprises a Fab or scFv. 
     
     
         138 . The method of any one of  claims 114  to  137 , wherein the protein is a modified Fc polypeptide that contains a non-native binding site capable of binding TfR. 
     
     
         139 . The method of any one of  claims 114  to  137 , wherein the protein comprises an antibody variable region that specifically binds TfR. 
     
     
         140 . The method of  claim 139 , wherein the protein comprises an antibody fragment. 
     
     
         141 . The method of  claim 140 , wherein the protein comprises a Fab or scFv. 
     
     
         142 . The method of any one of  claims 114  to  141 , wherein the protein linked to the agent is administered as part of a pharmaceutically acceptable carrier.